A hck inhibitor and a bcl-2 inhibitor for treating acute myeloid leukemia

ABSTRACT

This invention relates to compounds, compositions and methods for treating leukemia, such as acute myeloid leukemia, in subjects where one or more mutations in FLT3 kinase are present.

TECHNICAL FIELD

The present invention relates to compounds, compositions and methods fortreating leukemia, such as acute myeloid leukemia.

BACKGROUND ART

Over the last decade, leukemia stem cells (LSCs) have become recognizedas key players in human acute myeloid leukeumia (AML) pathogenesis aswell as chemotherapy resistance and relapse. The Src family kinase (SFK)hematopoietic cell kinase, or HCK, is overrepresented in primary humanAML LSCs in comparison to normal hematopoietic stem cells (HSCs). Theimportance of HCK and other SFKs, including LYN and FGR, in myeloidproliferation and differentiation has been demonstrated in knockoutmice. The myeloid-specific SFKs participate in wild-type and mutantkinase receptor (KIT) and fms-like tyrosine kinase 3 (FLT3) signalingand in the activation of the signal transducer and activator oftranscription 5 (STAT5). Myeloid-specific SFKs also are involved inextracellular signal-regulated kinase (ERK) pathways downstream ofbreakpoint cluster gene-Abelson murine leukemia fusion protein(BCR−ABL), and are involved in leukemogenesis in a mouse model ofBCR−ABL+B−ALL (acute lymphoblastic leukemia).

FLT3 is a type III receptor tyrosine kinase that plays important rolesin the differentiation and survival of hematopoietic stem cells in bonemarrow and has been observed to be over-expressed in AML and ALL. Avariety of gain-of-function mutations have been identified in these AMLpatients, such as FLT3−ITD (internal tandem duplication),FLT3−D835Y/E/V/H, and FLT3−K663Q, among which FLT3−ITD accounts forabout 30% of AML occurrence and is associated with poor prognosis. Themutated FLT3−ITD kinase promotes AML blast survival and proliferationthrough the downstream signaling mediators, including StatS, ERK andAKT. Therefore, FLT3−ITD kinase has been considered as a validated drugdiscovery target for FLT3−ITD positive AML.

A number of small molecule inhibitors have been reported to exhibitpotent FLT3 kinase inhibitory activities such as sunitinib, sorafenib,PKC412, CEP-701, UNC2025, MLN518, KW-2449 and AMG-925. In addition, therelatively more selective second generation FLT3 inhibitors such asAC2206, crenolanib and PLX3397 are being clinically evaluated and haveshown initial transient responses, but usually followed by quickdevelopment of resistance.

Mutations in genes such as NPM1, TET2, WT1, IDH1/2 and DNMT3A arecommonly found in AML, and recent work utilizing next-generationsequencing (NGS) suggests that certain mutations occur earlier thanothers based on variant allele frequencies (VAFs). Pre-leukemic cellsharboring early somatic mutations are thought to contribute toleukemogenesis and disease relapse. However, these mutations may not besufficient for leukemogenesis nor identify cells destined to becomemalignant.

Thus, there exists a need for elucidation of causational mutations forAML and effective treatments based on inhibiting or blocking aberrantpathways generated by those mutations. One effective combination thatcan provide durable remission of AML is provided in the combination of adual HCK/FLT3−ITD inhibitor and a BCL-2 inhibitor.

SUMMARY OF INVENTION

The present invention is summarized as follows.

1. A method of co-inhibiting HCK and BCL-2 in a cell, comprisingcontacting the cell with an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.

2. A method of killing a cell having an FLT3−ITD mutation, comprisingcontacting the cell with an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.

3. The method of item 1 or 2, further comprising contacting the cellwith an FLT3−ITD inhibitor.

4. The method of item 1 or 2, wherein the HCK inhibitor is a dualHCK/FLT3−ITD inhibitor.

5. A method of treating acute myeloid leukemia, comprising conjointlyadministering to a subject an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.

6. The method of item 5, wherein the subject has FLT3−ITD+ acute myeloidleukemia.

7. The method of item 5 or 6, wherein the subject has malignanthematopoiesis and/or non-malignant multilineage hematopoiesischaracterized by cells having one or more mutations in a gene selectedfrom DNMT3A, IDH2, IDH1, NPM1, TET2, CEBPA, ASXL1, EZH2, SETBP1, SMC3,KIT, NRAS, and WT1.

8. The method of any one of items 5-7, further comprising conjointlyadministering a FLT3−ITD inhibitor.

9. The method of any one of items 5-8, wherein the HCK inhibitor is adual HCK/FLT3−ITD inhibitor.

10. The method of item 8 or 9, wherein the HCK inhibitor, the FLT3−ITDinhibitor, and the BCL-2 inhibitor are administered simultaneously orsequentially in separate unit dosage forms.

11. The method of any one of items 5-10, comprising administering asingle unit dosage form comprising an HCK inhibitor, a BCL-2 inhibitor,and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

12. The method of item 11, wherein the single unit dosage form furthercomprises an FLT3−ITD inhibitor, or wherein the HCK inhibitor is a dualHCK/FLT3−ITD inhibitor.

13. The method of any preceding item, wherein the HCK inhibitor isselected from RK-20449, RK-20693, RK-24466, RK-20444, RK-20445, andRK-20466.

14. The method of any one of items 3, 4, or 7-12, wherein the FLT3−ITDinhibitor is selected from AC220, sorafenib, PKC412, CEP-701, UNC2025,MLN518, KW-2449, AMG-925, sunitinib, SU5614, AC2206, crenolanib, andPLX3397.

15. The method of any preceding item, wherein the BCL-2 inhibitor isselected from AT-101, TW-37, TM-1206, gossypolic acid, gossypolonicacid, apogossypol, apogossypolone, A385358, ABT-737, ABT-263, ABT-199,WEHI-539, BXI-61, BXI-72, obatoclax, JY-1-106, and SAHB peptides.

16. The method of item 15, wherein the BCL-2 inhibitor is selected fromgossypol, obatoclax, ABT-737, ABT-199, and ABT-263.

17. The method of item 16, wherein the BCL-2 inhibitor is ABT-199.

18. The method of item 17, wherein the HCK inhibitor is RK-20449 and theBCL-2 inhibitor is ABT-199.

19. The method of item 17, wherein the HCK inhibitor is RK-20693 and theBCL-2 inhibitor is ABT-199.

20. The method of item 17, wherein the FLT3−ITD inhibitor is AC220 andthe BCL-2 inhibitor is ABT-199.

21. The method of item 16, wherein the FLT3−ITD inhibitor is SU5614 andthe BCL-2 inhibitor is ABT-737.

22. The method of any preceding item, wherein the HCK inhibitor, and/orFLT3−ITD inhibitor, and/or the BCL-2 inhibitor is each present as apharmaceutically acceptable salt.

23. The method of any preceding item, wherein the HCK inhibitor, and/orFLT3−ITD inhibitor, and/or the BCL-2 inhibitor is each present in apharmaceutically acceptable composition.

Despite substantial clonal diversity defined by various combinations ofsomatic mutations, multi-kinase inhibition of SFKs and FLT3−ITDeffectively reduced AML in vivo, and the addition of BCL2 inhibitionsynergistically eliminated resistant AML. In certain embodiments, thecombined inhibition of anti-apoptotic and kinase signaling pathwaysthrough co-administration of RK-20449, a multi-kinase inhibitor ofFLT3−ITD and HCK, and ABT-199, a small molecule inhibitor of BCL-2, ledto successful elimination of human FLT3−ITD+AML in vivo despitesubstantial patient-to-patient heterogeneity and clonal diversity withinindividual patients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts mutational profiling of patient-derived subpopulationssorted by surface phenotype; the subpopulation undergoing NSGxenotransplantation to determine in vivo cell fate; and single-cellmutational profiling that was performed for patient-derivedsubpopulations with known cell fates and recipient-derived humanmultilineage hematopoietic cells and AML cells. FIG. 1A depictsmutational profiling of patient-derived subpopulations sorted by surfacephenotype. FIG. 1B depicts the sub-population undergoing NSGxenotransplantation to determine in vivo cell fate. If repopulation bymultilineage hematopoiesis occurred, the transplanted subpopulationcontained hematopoietic stem cells (HSCs); if xenotransplantationresulted in AML engraftment, the transplanted subpopulation containedleukemia-initiating cells (LICs). FIG. 1C depicts single-cell mutationalprofiling that was performed for patient-derived subpopulations withknown cell fates and recipient-derived human multilineage hematopoieticcells and AML cells.

FIG. 2-1 depicts identification of hematopoietic subpopulations in twopatients (patients 21 and 20).

FIG. 2-2 depicts identification of hematopoietic subpopulations in twopatients (patients 13 and 1). FIGS. 2A-2D depict identification ofhematopoietic subpopulations in four patients. In each patient sample, Tand B lymphoid populations and non-T non-B cells with distinct CD34 andCD38 surface expression were identified. Heat maps represent variantallele frequencies of NPM1, DNMT3A, CEBPA, IDH1, IDH2, TET2 and WT1genes in indicated subpopulations isolated from each patient. FLT3−ITDmutation was detected by PCR. FIG. 2A is patient 21; FIG. 2B is patient20; FIG. 2C is patient 13; and FIG. 2D is patient 1.

FIG. 3-1 depicts diverse subclones defined by various combinations ofsomatic mutations that were present in a patient-derivedleukemia-initiating population and a multilineage-engrafting population,and engrafted AML and multilineage human hematopoietic cells.

FIG. 3-2 depicts diverse subclones defined by various combinations ofsomatic mutations that were present in a patient-derivedleukemia-initiating population and a multilineage-engrafting population,and engrafted AML and multilineage human hematopoietic cells.

FIG. 3-3 depicts diverse subclones defined by various combinations ofsomatic mutations that were present in a patient-derivedleukemia-initiating population and a multilineage-engrafting population,and engrafted AML and multilineage human hematopoietic cells.Theleukemia-initiating population and the multilineage engraftingpopulation from four patients, isolated by surface phenotype andfunctionally defined by xenotransplantation, underwent single cellsequencing for indicated genes and single cell PCR for FLT3−ITD. Eachsingle cell is represented as a column of rectangles. The presence orabsence of mutations in each indicated gene is shown by colors of therectangles. FIG. 3A is patient 1; FIG. 3B is patient 21; FIG. 3C and 3Dis patient 13; and FIG. 3E is patient 20.

FIG. 4 depicts variant allele frequencies of nine genes in patient- andrecipient-derived AML cells in twelve AML cases represented as heatmaps. “1” refers to patient-derived AML cells; “1” and “2”, primary andsecondary recipient-derived AML cells, respectively. Blank squaresindicate that the sequence could not be read. Sequencing for FLT3 wasperformed to identify non−ITD FLT3 mutations. All patient-derived andrecipient-derived leukemia populations were positive for FLT3−ITD byPCR.

FIG. 5 depicts in vivo kinase inhibition that induces apoptosis ofFLT3−ITD+AML cells with various combinations of co-existing somaticmutations and enhances dependence on Bcl-2 for survival. Each patientcase was classified by in vivo RK-20449 treatment responses ofrecipients as follows: Complete responder (FIG. 5A), if all recipientstreated showed residual BM human CD45+chimerism <5%; good responder(FIG. 5B), if the case did not meet the criterion for complete responderbut all recipients showed <50% residual BM human CD45+; partialresponder (FIG. 5C), if at least one of the recipients showed greaterthan 50% residual BM human CD45+. PB time-course of human AML chimerism(leftmost panels) for RK-20449 treated recipients and BM (middle panels)and spleen (rightmost panels) human AML chimerism at the time ofsacrifice of RK-20449 treated and untreated recipients are shown.Pre-treatment PB human AML cell chimerism is shown at week 0. Thenumbers of recipients for each patient/each treatment group andpre-/post-treatment AML chimerisms are shown in FIG. 8.

FIG. 6A depicts the time courses of PB responses to in vivo treatmentsfor four treatment groups (no treatment, ABT-199 alone, RK-20449 aloneand RK-20449 and ABT-199). Recipients were phlebotomized weekly withtime 0 representing pretreatment PB human CD45+AML chimerism. Final BMand spleen human AML chimerisms for nine cases that showed completeresponses to combined treatment with RK-20449 and ABT-199.

FIG. 6B depicts human AML cell chimerisms in BM and spleen following invivo treatment (no treatment, ABT-199 alone, RK-20449 alone,combination) for AML cases that showed complete responses. Each circlerepresents an AML-engrafted recipient.

FIG. 6C depicts that by serial transplantation, residual humanAML-initiation capacity in human CD45+cells following in vivo treatmentwas assessed for four treatment groups. The transplanted AML cells wereisolated from 2.5% of viable residual human AML cells remaining inrecipients of each treatment group into each secondary recipient. Meanand SEM for human CD45+AML cell chimerism in the BM of secondaryrecipients are shown. Each circle represents a secondary recipient.

FIG. 6D depicts a schematic representation of the relationship betweenFLT3−ITD and other AML-associated mutations. A pre-leukemic cellpopulation consists of HSCs carrying mutations in genes such as DNMT3A,TET2 and CEBPA in various combinations. These permission mutations maycooperate with FLT3−ITD for malignant transformation but do not precludemultilineage maturation to lymphoid and myeloid cells. On the otherhand, FLT3−ITD possesses the greatest malignant potential inFLT3−ITD+AML patients. Acquisition of FLT3−ITD at the level of HSCsresults in loss of multilineage differentiation capacity, convertingpre-leukemic HSCs into LSCs. More mature progenitors and differentiatedmyeloid cells may also acquire FLT3−ITD and become LICs.

FIG. 7 provides clinical characteristics of patients. M0, M1, M2, M4,M5, M5a that indicated French-American-British classification atdiagnosis. MDS, myelodysplastic syndrome; AML/MRC, acute myeloidleukemia with myelodysplasia-related changes; MF, myelofibrosis; CMML,chronic myelomonocytic leukemia; HSCT, hematopoietic stem celltransplantation; PB, peripheral blood; BM, bone marrow; IDA, idarubicin;AraC, cytarabine; uCBT, umbilical cord blood transplantation; CR,complete response; MIT, mitoxanthrone; GVHD, graft-versus-host disease;PBSCT, mobilized peripheral blood stem cell transplantation; AZA,azacytidine; HiDAC, high-dose cytarabine; CNS, central nervous system;GO, gemtuzumab ozogamicin; MUD, matched unrelated donor; BMT bone marrowtransplantation; MRD, matched related donor.

FIG. 8 provides human AML chimerisms in PB, BM and spleen ofAML-engrafted recipients treated with RK-20449 alone. The percent humanCD45+cells in pre- and post-treatment PB and post-treatment BM andspleen are shown for recipients engrafted with human AML. PB, peripheralblood; BM, bone marrow; n, number of recipients; SEM, standard error ofthe mean; na, data not available. Response groups: Complete,post-treatment BM human CD45+<5% in every recipient treated; Good,post-treatment BM human CD45+<5% in all but one recipient treated;Partial, post-treatment BM human CD45+ is statistically significantlyreduced compared with untreated group but >50% in one recipient treated.

FIG. 9 provides human AML chimerisms in PB, BM and spleen ofAML-engrafted recipients treated with ABT-199 alone, RK-20449 alone orin combination. The percent human CD45+cells in pre- and post-treatmentPB and post-treatment BM and spleen are shown for recipients engraftedwith human AML. Untreated and RK-20449-treated recipient data areduplicated from FIG. 8 as comparison. Response groups: Complete,post-treatment BM human CD45+<5% in every recipient treated; Good,post-treatment BM human CD45+<5% in all but one recipient treated.

FIG. 10 provides statistics comparing pre- and post-treatment PB humanAML chimerisms in recipients treated with ABT-199 alone, RK-20449 aloneor in combination. The mean+/−s.e.m. of percent human CD45+cells inperipheral blood of recipients engrafted with human AML is shown. BM,number of recipients; p, paired two-tailed t-test comparing pre- andpost-treatment PB human % CD45+; na, not powered to detect statisticallysignificant differences.

FIG. 11 depicts statistics comparing BM and spleen human AML chimerismsin recipients treated with ABT-199 alone, RK-20449 alone or incombination. The mean+/−s.e.m. of percent human CD45+cells in recipientsengrafted with human AML is shown. BM, bone marrow; n, number ofrecipients; p, unpaired two-tailed t-test between indicated parings; na,not powered to detect statistically significant differences.

DESCRIPTION OF EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art ofthe present disclosure. The following references provide one of skillwith a general definition of many of the terms used in this disclosure:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “ includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used herein, the term “detect” refers to identifying the presence,absence or amount of the analyte to be detected. The terms “eliminate”or “elimination” refer to an absence of analyte being detected. One ofordinary skill in the art readily appreciates that measurement methodsinherently possess a limit(s) to its lowest and highest levels ofdetection. Thus, an indication of not detected as used herein is not tobe construed to mean the analyte is not present at all. It is simply notpresent between the upper or lower limits of the detection method.

By “effective amount” or “therapeutically effective amount” is meant theamount of an active agent required to ameliorate the symptoms of adisease relative to an untreated subject. The effective amount of activeagent(s) disclosed herein for therapeutic treatment of a disease variesdepending upon a number of factors, including, but not limited to, themanner of administration, the age, body weight, and general health ofthe subject. The attending physician or veterinarian can decide theappropriate amount and dosage regimen.

The term “subject,” “patient” or “individual” to which administration iscontemplated includes, but is not limited to, humans (i.e., a male orfemale of any age group, e.g., a pediatric subject (e.g., infant, child,adolescent) or adult subject (e.g., young adult, middle-aged adult orsenior adult)) and/or other primates (e.g., cynomolgus monkeys, rhesusmonkeys); mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds,including commercially relevant birds such as chickens, ducks, geese,quail, and/or turkeys.

As used herein, a treatment that “prevents” a disorder or conditionrefers to a treatment that, in a statistical sample, reduces theoccurrence or frequency of the disorder or condition in the treatedsample relative to an untreated control sample, or delays the onset orreduces the severity of one or more symptoms of the disorder orcondition relative to the untreated control sample. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic compounds suchthat the second compound is administered while the previouslyadministered therapeutic compound is still effective in the body (e.g.,the two compounds are simultaneously effective in the patient, which mayinclude synergistic effects of the two compounds). For example, thedifferent therapeutic compounds can be administered either in the sameformulation or in a separate formulation, either concomitantly orsequentially. In certain embodiments, the different therapeuticcompounds can be administered within one hour, 12 hours, 24 hours, 36hours, 48 hours, 72 hours, or a week of one another. Thus, an individualwho receives such treatment can benefit from a combined effect ofdifferent therapeutic compounds.

Overview

Despite substantial clonal diversity defined by various combinations ofsomatic mutations, multi-kinase inhibition of SFKs and FLT3−ITDeffectively reduced AML in vivo, and the addition of BCL2 inhibitionsynergistically eliminated resistant AML. In certain embodiments, thecombined inhibition of anti-apoptotic and kinase signaling pathwaysthrough co-administration of RK-20449, a multi-kinase inhibitor ofFLT3−ITD and HCK, and ABT-199, a small molecule inhibitor of BCL-2, ledto successful elimination of human FLT3−ITD+AML in vivo despitesubstantial patient-to-patient heterogeneity and clonal diversity withinindividual patients.

By integrating single cell genomics with in vivo functional assessment,patient-specific mutational profiles were identified that defined clonalarchitectures in preleukemic stem cells and leukemia-initiating cells inFLT3−ITD+AML. Among the mutations, FLT3−ITD possessed the greatestmalignant potential in vivo, demarcating the transition frompre-leukemia to leukemia. Despite diverse co-existing mutations,RK-20449 effectively decreased the leukemic cell population in aPDX-model to below the limit of detection such as less than 0.01%remaining AML cells using flow cytometry and immunohistochemistry usingmultiple monoclonal antibodies. RK-20449 also enhanced BCL-2 dependencefor leukemia cell survival, and co-inhibition of BCL-2 decreased thehuman leukemia cell count in vivo to below the limit of detection.Co-inhibition of these two pathways for malignant transformation andtumor survival is successful despite complex patient-specific mutationallandscapes.

Further, specific inhibition of HCK can increase the potency of thecombination of the FLT3−ITD inhibitors and BCL2 inhibitors describedherein. HCK is a member of the Src-family of non-receptor tyrosinekinases, which plays many roles in signaling pathways involved in theregulation of cell processes. HCK is expressed in cells of hematopoieticorigin, specifically myelomonocytic cells and B lymphocytes. Itparticipates in phagocytosis, adhesion, migration, regulation ofprotrusion formation on cell membrane, lysosome exocytosis, podosomeformation and actin polymerization. High levels of HCK are present inchronic myeloid leukemia and other hematologic tumors. HCK could alsoplay a role in the genesis of acute myeloid leukemia.

In certain embodiments, provided herein is a method of co-inhibiting HCKand BCL-2 in a cell, comprising contacting the cell with an HCKinhibitor and a BCL-2 inhibitor, or a pharmaceutically acceptablecomposition thereof. In certain preferred embodiments, the methodfurther comprises contacting the cell with a FLT3−ITD inhibitor, such asa dual HCK/FLT3−ITD inhibitor. In certain embodiments the cell is invitro. In other embodiments, the cell is in vivo. In other embodiments,the cell is an acute myeloid leukemic cell.

In certain embodiments, provided herein is a method of killing a cellhaving a FLT3−ITD mutation, comprising contacting the cell with an HCKinhibitor and a BCL-2 inhibitor, or a pharmaceutically acceptablecomposition thereof. In certain preferred embodiments, the methodfurther comprises contacting the cell with a FLT3−ITD inhibitor, such asa dual HCK/FLT3−ITD inhibitor. In certain embodiments the cell is invitro. In other embodiments, the cell is in vivo. In other embodiments,the cell is an acute myeloid leukemic cell.

In certain embodiments, provided herein is a method of treating acutemyeloid leukemia, comprising conjointly administering to a subject anHCK inhibitor and a BCL-2 inhibitor, or a pharmaceutically acceptablecomposition thereof. In certain preferred embodiments, the methodfurther comprises conjointly administering to a subject a FLT3−ITDinhibitor. In other preferred embodiments, the HCK inhibitor is a dualHCK/FLT3−ITD inhibitor. Other embodiments provide a compositioncomprising a therapeutically effective amount of an HCK inhibitor and aBCL-2 inhibitor, or a pharmaceutically acceptable composition thereof,for the treatment of acute myeloid leukemia. In certain preferredembodiments, the composition further comprises a therapeuticallyeffective amount of a FLT3−ITD inhibitor. In other preferredembodiments, the HCK inhibitor is a dual HCK/FLT3−ITD inhibitor. Inother embodiments, provided herein is the use of a therapeuticallyeffective amount of an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof, in the manufacture of amedicament for the treatment of acute myeloid leukemia. In certainpreferred embodiments, the use further comprises a therapeuticallyeffective amount of a FLT3−ITD inhibitor, while in other preferredembodiments, the HCK inhibitor is a dual HCK/FLT3−ITD inhibitor. Inrelated embodiments, the invention provides the use of a therapeuticallyeffective amount of an HCK inhibitor or a BCL-2 inhibitor in themanufacture of a medicament for use in a method of treating acutemyeloid leukemia by the conjoint administration of an HCK inhibitor anda BCL-2 inhibitor. In certain preferred embodiments, the method furthercomprises conjointly administering a therapeutically effective amount ofa FLT3−ITD inhibitor, while in other preferred embodiments, the HCKinhibitor is a dual HCK/FLT3−ITD inhibitor.

In certain embodiments, provided herein is a method of co-inhibitingHCK, FLT3−ITD, and BCL-2 in a cell, comprising contacting the cell withan HCK inhibitor, a FLT3−ITD inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof. In some embodiments,the HCK inhibitor and the FLT3−ITD inhibitor are provided as a dualHCK/FLT3−ITD inhibitor. In certain embodiments the cell is in vitro. Inother embodiments, the cell is in vivo. In certain such embodiments, thecell is an acute myeloid leukemic cell.

In certain embodiments, provided herein is a method of killing a cellhaving an FLT3−ITD mutation, comprising contacting the cell with an HCKinhibitor, a FLT3−ITD inhibitor, and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof. In some embodiments,the HCK inhibitor and the FLT3−ITD inhibitor are provided as a dualHCK/FLT3−ITD inhibitor. In certain embodiments the cell is in vitro. Inother embodiments, the cell is in vivo. In certain such embodiments, thecell is an acute myeloid leukemic cell.

In certain embodiments, provided herein is a method of treating acutemyeloid leukemia, comprising conjointly administering to a subject anHCK inhibitor, a FLT3−ITD inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof. In some embodiments,the HCK inhibitor and the FLT3−ITD inhibitor are provided as a dualHCK/FLT3−ITD inhibitor. Other embodiments provide composition comprisinga therapeutically effective amount of an HCK inhibitor, a FLT3−ITDinhibitor and a BCL-2 inhibitor, or a pharmaceutically acceptablecomposition thereof, for the treatment of acute myeloid leukemia. Insome embodiments, the HCK inhibitor and the FLT3−ITD inhibitor areprovided as a dual HCK/FLT3−ITD inhibitor. In other embodiments,provided herein is the use of a therapeutically effective amount of anHCK inhibitor, a FLT3−ITD inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof, in the manufacture of amedicament for the treatment of acute myeloid leukemia. In someembodiments, the HCK inhibitor and the FLT3−ITD inhibitor are providedas a dual HCK/FLT3−ITD inhibitor. In related embodiments, the inventionrelates to the use of an HCK inhibitor, a FLT3−ITD inhibitor or a BCL-2inhibitor in the manufacture of a medicament for treating acute myeloidleukemia in a method comprising the conjoint administration of the HCKinhibitor, the FLT3−ITD inhibitor or the BCL-2 inhibitor with the otherof the HCK inhibitor, FLT3−ITD inhibitor and the BCL-2 inhibitor.

In certain embodiments, the HCK inhibitor is selected from RK-20449,RK-20693, RK-24466, RK-20444, RK-20445, and RK-20466. In otherembodiments, the HCK inhibitor is selected from RK-20449, RK-20693,RK-24466, RK-20444, RK-20445, RK-20466, RK-20730, RK-20690, RK-20781,RK-20786, RK-20888, RK-20658, RK-20686, RK-20696, RK-20709, RK-20721,RK-20694, RK-20703, RK-20718, RK-20744, and compounds having HCKinhibitory activity disclosed in WO2014/017659.

In some embodiments, the FLT3−ITD inhibitor is selected from AC220,sorafenib, PKC412, CEP-701, UNC2025, MLN518, KW-2449 and AMG-925,sunitinib, SU5614, AC2206, crenolanib, and PLX3397. In certain preferredembodiments, the HCK inhibitor is RK-20449. In other embodiments, theFLT3−ITD inhibitor is AC220.

In other embodiments, the BCL-2 inhibitor is selected from AT-101,TW-37, TM-1206, gossypol, gossypolic acid, gossypolonic acid,apogossypol, apogossypolone, A385358, ABT-737, ABT-263, ABT-199,WEHI-539, BXI-61, BXI-72, obatoclax, ONT-701, S1, JY-1-106, and SAHB(stabilized α\och-helix of Bcl-2 domains) peptides. In certainembodiments, the BCL-2 inhibitor is selected from gossypol, obatoclax,ABT-737, ABT-199, and ABT-263. In certain preferred embodiments, theBCL-2 inhibitor is ABT-199.

Other BCL-2 inhibitors contemplated herein include, but are not limitedto, those disclosed in Anderson, et al. Seminars in Hematology 201451:219-227; Bajwa et al. Expert Opin Ther. Pat. 2012 22:37-55;Oltersdorf, T., et al. Nature 2005 435:677-681; Tse, C. et al. CancerResearch 2008 68:3421-3428; Souers, A. J. et al. Nature Medicine 201319:202-208; Nguyen, M. et al. PNAS 2007 104:19512-19517; Zhang, A. etal. Int'l J. Cancer 2011 128:1724-1735; and Wei, J. et al. J. Med. Chem.2009 52:4511-4523.

In certain preferred embodiments, the HCK inhibitor is RK-20449 and theBCL-2 inhibitor is ABT-199. In other embodiments, the HCK inhibitor isRK-20693 and the BCL-2 inhibitor is ABT-199. In other embodiments, theFLT3−ITD inhibitor is AC220 and the BCL-2 inhibitor is ABT-199. In otherembodiments, the FLT3−ITD inhibitor is SU5614 and the BCL-2 inhibitor isABT-737.

In certain embodiments, the effective concentration to inhibit HCK isgreater than the effective concentration to inhibit BCL-2. In otherembodiments, the effective concentration to inhibit HCK is less than theeffective concentration to inhibit BCL-2. In other embodiments, theeffective concentration to inhibit FLT3−ITD and the effectiveconcentration to inhibit HCK are substantially similar.

In certain embodiments, the effective concentration to inhibit FLT3−ITDis greater than the effective concentration to inhibit BCL-2. In otherembodiments, the effective concentration to inhibit FLT3−ITD is lessthan the effective concentration to inhibit BCL-2. In other embodiments,the effective concentration to inhibit FLT3−ITD and the effectiveconcentration to inhibit BCL-2 are substantially similar.

In certain embodiments, the subject has malignant hematopoiesis and/ornon-malignant multilineage hematopoiesis characterized by cells havingone or more mutations in a gene selected from DNMT3A, IDH2, IDH1, NPM1,TET2, CEBPA, ASXL1, EZH2, SETBP1, SMC3, KIT, NRAS, and WT1.

Combination Therapies

The invention generally relates to conjoint therapies involving a HCKinhibitor and a BCL-2 inhibitor, optionally with a FLT3−ITD inhibitor.In certain embodiments of the invention, the therapeutically effectiveamount of the FLT3−ITD and/or HCK inhibitor is less than itstherapeutically effective amount would be where the BCL-2 inhibitor isnot administered. Similarly, the therapeutically effective amount of theBCL-2 inhibitor may be less than its therapeutically effective amountwould be where the FLT3−ITD inhibitor and/or HCK inhibitor is notadministered, and the therapeutically effective amount of the HCKinhibitor may be less than its therapeutically effective amount would bewhere the FLT3−ITD inhibitor and/or BCL-2 inhibitor is not administered.In this way, undesired side effects associated with high doses of eitheragent may be minimized. Other potential advantages (including withoutlimitation improved dosing regimens and/or reduced drug cost) will beapparent to those of skill in the art. The FLT3−ITD inhibitor, HCKinhibitor, and/or BCL-2 inhibitor may be administered separately fromeach other, e.g., as part of a multiple dose regimen. Alternatively, twoor more inhibitors may be part of a single dosage form, e.g., mixedtogether in a single composition.

The amount of any of the FLT3−ITD, HCK, and BCL-2 inhibitors that can becombined with the carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. Preferably, compositions of this invention should beformulated so that a dosage of between about 0.01 - about 100 mg/kg bodyweight/day of an HCK inhibitor, a dosage of between about 0.01 - about100 mg/kg body weight/day of a FLT3−ITD inhibitor, and a dosage ofbetween about 0.01 - about 100 mg/kg body weight/day of a BCL-2inhibitor can be administered. Also preferably, compositions of thisinvention should be formulated so that a dosage of between about 0.01 -about 100 mg/kg body weight/day of an HCK inhibitor and a dosage ofbetween about 0.01 - about 100 mg/kg body weight/day of a BCL-2inhibitor can be administered. In certain embodiments, a therapeuticallyeffective amount of a dual HCK/FLT3 inhibitor or a BCL-2 inhibitordescribed herein may be administered alone or in combination withtherapeutically effective amounts of other compounds useful for treatingAML. In certain embodiments, a therapeutically effective amount of anHCK inhibitor, a FLT3−ITD inhibitor or a BCL-2 inhibitor describedherein may be administered alone or in combination with therapeuticallyeffective amounts of other compounds useful for treating AML.

Compositions and Salts

In some embodiments, disclosed compounds can be in the form of apharmaceutically acceptable composition. Disclosed herein arepharmaceutical compositions comprising a FLT3−ITD inhibitor, and/or HCKinhibitor, and/or a BCL-2 inhibitor as described herein and apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-poly-oxypropylene-block polymers,polyethylene glycol and wool fat. Other pharmaceutically acceptablecarriers, adjuvants or vehicles include water, saline anddimethyl-sulfoxide, as well as other hydrophobic or hydrophilicsolvents.

Examples of suitable aqueous and nonaqueous carriers which can beemployed in pharmaceutical compositions include water, ethanol, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents, dispersing agents, lubricants,and/or antioxidants. Prevention of the action of microorganisms upon thecompounds described herein can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It can also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound described herein with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation a compound as disclosed herein with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Preparations for such pharmaceutical compositions are well-known in theart. See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, WilliamG, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill,2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition,Churchill Livingston, New York, 1990; Katzung, ed., Basic and ClinicalPharmacology, Ninth Edition, McGraw Hill, 2003; Goodman and Gilman,eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGrawHill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., LippincottWilliams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia,Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all ofwhich are incorporated by reference herein in their entirety. Exceptinsofar as any conventional excipient medium is incompatible with thecompounds provided herein, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutically acceptable composition,the excipient's use is contemplated to be within the scope of thisdisclosure.

Provided herein are pharmaceutically acceptable salts which refer tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subjects without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptablesalts of the compounds provided herein include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. In some embodiments, organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid,maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, andthe like.

The salts can be prepared in situ during the isolation and purificationof the disclosed compounds, or separately, such as by reacting the freebase or free acid of the compound with a suitable base or acid,respectively. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN+(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases fromwhich salts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines, including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. In some embodiments,the pharmaceutically acceptable base addition salt is chosen fromammonium, potassium, sodium, calcium, and magnesium salts.

Administration

Compositions of the present invention may be administered orally,parenterally (including subcutaneous, intramuscular, intravenous andintradermal), by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. In some embodiments,provided compounds or compositions are administrable intravenouslyand/or intraperitonally. In certain preferred embodiments, the disclosedmethods include orally or parenterally administering any two of, or allthree of, a FLT3−ITD inhibitor, an HCK inhibitor, and a BCL-2 inhibitor.

The term “parenteral,” as used herein, includes subcutaneous,intravenous, intramuscular, intraocular, intravitreal, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic,intraperitoneal, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,subcutaneously, intraperitoneally or intravenously. Sterile injectableforms of the compositions of this invention may be aqueous or oleaginoussuspension. These suspensions may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In some embodiments, aprovided oral formulation is formulated for immediate release orsustained/delayed release. In some embodiments, the composition issuitable for buccal or sublingual administration, including tablets,lozenges and pastilles. A compound disclosed herein can also be inmicro-encapsulated form.

The amount of a compound of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the subject being treated and theparticular mode of administration. In certain embodiments, providedcompositions should be formulated so that a dosage of between about 0.01to about 100 mg/kg body weight/day of the compound can be administeredto a subject receiving these compositions. In other embodiments, thedosage is from about 0.5 to about 100 mg/kg of body weight, or betweenabout 1 mg and about 1000 mg/dose, about every 4 to 120 hours, oraccording to the requirements of the particular drug. Typically, thepharmaceutical compositions of this invention will be administered fromabout 1 to about 6 times per day.

In some embodiments, the compound is formulated for oral administrationat a dosage of approximately 5 mg/kg to approximately 10 mg/kg,preferably at a dosage of approximately 7.5 mg/kg.

It should also be understood that a specific dosage and treatmentregimen for any particular subject will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Subjects may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.In case of conflict, the present application, including any definitionsherein, will control.

Examples

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions withart-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and Description of how tomake and use the methods of the invention, and are not intended to limitthe scope of what the inventor(s) regard(s) as the invention.

Unless noted otherwise, the starting materials for the experimentsdescribed herein were obtained from commercial sources or knownprocedures and were used without further modification.

General Methods Compounds

The following compounds can be synthesized according to, e.g., themethods disclosed in WO2014/017659.

RK-20449 (also known as A 419259):

7-((1R,4R)-4-(4-methylpiperazin-l-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20693:

N-(4-(4-amino-7-(trans-4-(4-methylpiperazin-1-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-3-phenylpropanamide

RK-24466:

7-c yclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20444:

1-(4-(4-amino-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-3-benzylurea

RK-20445:

N-(4-(4-amino-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)benzamide

RK-20466:

7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20730:

N-(4-(4-amino-7-((1R,4R)-4-(3-methyl-5,6-dihydro-[1,2,4] triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20690:

7-41R,4R)-4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20781:

7-(1-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)piperidin-4-yl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20888:

1-((1S,4S)-4-((8-methyl-8-azabicyclo[3.2.1]octan-3-yl)amino)cyclohexyl)-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

RK-20658:

5-(4-phenoxyphenyl)-7-((1r,4r)-4-(piperazin-1-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20686:

7-((1R,4R)-4-(3-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK20696:

5-(4-phenoxyphenyl)-7-((1S,4S)-4-((pyridin-3-ylmethyl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20709:

7-([1,4′-bipiperidin]-4-yl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20721:

2-(((1R,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)aceticacid trihydrochloride

RK-20694:

5-(4-phenoxyphenyl)-7-((1S,4S-4-((pyridin-4-ylmethyl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20703:

5-(4-phenoxyphenyl)-7-((1S,4S)-4-((tetrahydro-2H-pyran-4-yl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20718:

2-(((1S,4S)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)aceticacid trihydrochloride

RK-20719:

2-(((1S ,4S)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)acetamide

RK-20722:

2-(((1R,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)acetamide

RK-20752:

N-((1S,4S)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)isonicotinamide

RK-20952:

7-((1R,4R)-4-(4-(tert-butyl)piperazin-1-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20618:

N-(4-(4-amino-1-((1R,4R)-4-(4-methylpiperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20725:

1-((1R,4R)-4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)cyclohexyl)-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

RK-20729:

N-(4-(4-amino-7((1S,4S)-4-(3-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20732:

N-(4-(4-amino-7-((1r,4r)-4-(4-methylpiperazin-1-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-1-methyl-1H-indole-2-carboxamide

RK-20746:

N-((1R,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)picolinamide

RK-20755:

N-((1R,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)nicotinamide

RK-20768:

N-((1R,4R)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)isonicotinamide

RK20770:

N-(4-(4-amino-1-((1r,4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)picolinamide

RK-20775:

N-((1R,4R)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)nicotinamide

RK-20777:

N-((1R,4R)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)picolinamide

RK-20791:

N-(4-(4-amino-1((1R,4R)-4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-1-methyl-1H-indole-2-carboxamide

RK-20798:

N-(4-(4-amino-1-(1-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-1-methyl-1H-indole-2-carboxamide

RK-20819:

N-(4-(4-amino-7-((1S,4S)-4-(4-methylpiperazin-1-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20820:

N-(4-(4-amino-7-((1R,4R)-4-(4-methylpiperazin-1-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20824:

N-(4-(4-amino-1-((1R,4R)-4-(4-methylpiperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-1-methyl-1H-indole-2-carboxamide

RK-20826:

N-(4-(4-amino-1-((1R,4R)-4-(3-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20901:

N-(4-(4-amino-7-((1R,4R)-4-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20908:

N-(4-(4-amino-7-((1s,4s)-4-(3-ethyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20909:

N-(4-(4-amino-7-((1r,4r)-4-(3-ethyl-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20918:

N-(4-(4-amino-7-((1s,4s)-4-(3-isopropyl-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20919:

N-(4-(4-amino-7-((1R,4R)-4-(3-isopropyl-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20920:

N-(4-(4-amino-7-((1S,4S)-4-(3-methyl-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20921:

N-(4-(4-amino-7-((1R,4R)-4-(3-methyl-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20930:

N-(4-(4-amino-7-((1S,4S)-4-(2-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20932:

N-(4-(4-amino-7-((1S,4S)-4-(6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)cyclohexy1)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20942:

N-(4-(4-amino-7-((1R,4R)-4-(3-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide

RK-20627:

7-((1R,4R)-4-(4-(2-methoxyethyl)piperazin-1-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20629:

2-(4-((1R,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)piperazin-1-yl)ethanol

RK-20640:

7-((1R,4R)-4-(4-isopropylpiperazin-1-yl)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20695:

5-(4-phenoxyphenyl)-7-((1R,4R)-4-((pyridin-4-ylmethyl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20697:

5-(4-phenoxyphenyl)-7-((1R,4R)-4-((pyridin-3-ylmethyl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20698:

5-(4-phenoxyphenyl)-7-((1S,4S)-4-((pyridin-2-ylmethyl)amino)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20710:

7-((1S,4S)-4-(((1-methyl-1H-pyrazol-5-yl)methyl)amino)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20711:

7-((1R,4R)-4-(((1-methyl-1H-pyrazol-5-yl)methyl)amino)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20712:

7-((1S,4S)-4-(((1-methyl-1H-pyrazol-3-yl)methyl)amino)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20724:

1-((1S,4S)-4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)cyclohexyl)-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

RK-20733:

(S)-3-(((1S,4R)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)pyrrolidin-2-one

RK-20734:

(S)-3-(((1R,4S)-4-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)amino)pyrrolidin-2-one

RK-20758:

7-((1S,4S)-4-((8-methyl-8-azabicyclo[3.2.1]octan-3-yl)amino)cyclohexyl)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20898:

5-(4-phenoxyphenyl)-7-((1R,4R)-4-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)cyclohexyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

RK-20620:

1-((1R,4R)-4-(4-methylpiperazin-1-yl)cyclohexyl)-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The following compounds are commercially available:

AC220 (also known as quizartinib):

Urea, N-

[5-(1,1-dimethylethyl)-3-isoxazolyl]-N′-[4-[7-[2-(4-morpholinyl)ethoxy]imidazo[2,1-b]benzothiazol-2-yl]phenyl]-.

ABT-199 (also known as venetoclax):

4-[4-[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[3-nitro-4-[[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-benzamide

Human Samples

All experiments were performed with authorization from the InstitutionalReview Board for Human Research at RIKEN and Toranomon Hospital. Allpatient samples were collected at Toranomon Hospital with writteninformed consent. CB samples were purchased from Lonza.

Mice

NOD.Cg-PrkdcscidIl2rgtmlWjl/Sz (NSG) mice were developed at The JacksonLaboratory (Shultz LD et al., J Immunol 2005, Ishikawa F et al., Blood2005) . Mice were bred and maintained under defined flora at the animalfacility of RIKEN and at The Jackson Laboratory according to guidelinesestablished by at the Institutional Animal Committees at eachinstitution.

Flow Cytometry

The following monoclonal antibodies (mAbs) were used for flow cytometry:Mouse anti-human CD19, CD3, CD33, CD34, CD38, CD4, and CD45; Ratanti-mouse Ter119 and CD45 (BD Biosciences). Analyses were performedwith FACSAriaIII and FAC-SCantoII (BD). To obtain cells for xenogeneictransplantation, BV786-conjugated anti-CD3 mAb, BV605-conjugatedanti-CD19 mAb, BV421-conjugated anti-CD33 mAb, PE-Cy7-conjugaedanti-CD34 mAb, APC-conjugated anti-CD38 mAb, FITC-conjutaged anti-CD90mAb, and PE-conjugated anti-CD45RA mAb were used. For single cellsorting, 100mum nozzle was used.

Transplantation

NSG newborns received 1.5Gy total body irradiation followed byintravenous injection of purified human cells. Donor cells were purifiedaccording to their cell surface phenotype using monoclonal antibodiesagainst human CD34, CD38, CD90, CD45RA, CD3, CD19 and CD33. Engraftmentlevels of human cells in the NSG recipients were assessed byretro-orbital phlebotomy and flow cytometry.

Genome Analysis

DNA was extracted from human cells purified from patient samples orrecipient organs using DNeasy Blood & Tissue Kit (QIAGEN). PCR detectionof FLT3−ITD was performed using TaKaRa PCR FLT3/ITD Mutation DetectionSet (Takara). The DNA sequences in bulk were determined bynext-generation DNA sequencing (NGS). After fragmented with a Covaris5220 (Covaris Inc., Wobum, Mass. USA), the fragmented genomic DNA (10ng) was converted to NGS sequencing library with a KAPA Hyper Prep Kit(KAPA Biosystems, Wilmington, Mass.) according to protocol provided bythe supplier. Targeted sequencing of AML-related genes was carried outby a hybridization-capture method with xGen AML Cancer Panel v1.0(Integrated DNA Technologies, Inc., Coralville, Iowa) according toprotocol provided by the supplier. The hybridization-captured DNAlibrary was subjected to NGS in a paired-end read mode (600 cycles) withan Illumina Miseq (Illumina, Inc., San Diego, Calif.). The obtained DNAsequences were mapped onto human genome sequence (hg19) using BWA, thenrealigned with a Realigner Target Creator in Geome Analysis Toolkit.After treatment with Fix Mate Information in Picard and Quality ScoreCovariate and Table Recalibration in Genome Analysis Toolkit (v.1.6-13),variants were detected with VarScan.

Single-cell variation analysis was carried out for single cells sortedon a BD FACS Aria into 96-well plates. After single-cell genomeamplification with a MALBAC Single Cell WGA Kit (Yikon Genomics, Beijin,China), target regions of genes of interest were PCR-amplified withprimers including well indexes by PCR as described in SupplementaryInformation. The PCR products were sequenced in a paired-end read mode(600 cycles) on an Illumina Miseq. The obtained DNA sequences weremapped onto human genome sequence (hg 19) with BWA (v0.7.12) and mergedpaired-end reads with SAMtools. Variant detection and frequencycalculation were done with mpileup in SAMtools.

In Vivo Treatment

In vivo treatment experiments were performed with AML-engrafted NSGrecipients using RK-20449 (Ref. STM 2013) and ABT-199 (Souers A J etal., Nature Medicine 2013, Pan R et al., Cancer Discovery 2014). Therecipients were administered RK-20449 intraperitoneally 30 mg/kg twice aday, ABT-199 orally 70 mg/kg once a day or both RK-20449 and ABT-199 atthese dosing schedules. The mice were sacrificed when they becamemoribund or after 4˜6 weeks of treatment, and human AML chimerisms inBM, spleen and PB were determined using flow cytometry. In secondarytransplantation, each mouse received 7-AAD(-) viable human CD45+cellsfrom 2.5% of total BM that remained in AML-engrafted recipients at thetime of sacrifice, to simulate relapse occurring from residual viableAML cells. All treated recipients and their pre- and post-treatmentengraftment data are tabulated in FIGS. 8 and 9.

Statistical Analysis

Numerical data are presented as mean+/−SEM. The differences wereexamined with two-tailed t test (GraphPad Prism, GraphPad).

EXAMPLES Example 1

Evaluation of FLT3−ITD+leukemic subclones

Bone marrow (BM) or peripheral blood (PB) samples were obtained from 23patients with FLT3−ITD+AML, whose diagnosis, clinical course andclinical outcomes are detailed in FIG. 7. The majority of patients hadpoor prognostic factors such as complex chromosomal abnormalities inaddition to FLT3−ITD mutation and/or had known aggressive disease(induction failure, relapse after multiple stem cell transplantations).

To evaluate the contribution of AML-associated somatic mutations toleukemogenesis by linking mutations with in vivo function, the followingexperiments performed: 1) Population-level mutation screening in surfacephenotype-defined hematopoietic cell subpopulations isolated frompatients, 2) Functional assessment of the in vivo fates of thosesubpopulations by NSG xenotransplantation, and 3) Single cell sequencingto track patient-derived clones in xenotransplantation recipients (see,FIGS. 1A, 1B, and 1C).

In normal human hematopoiesis, HSC-enriched CD34+CD38−cells repopulateNSG recipients. Within this population, CD34+CD38−(CD90−)CD45RA−cellsare capable of multilineage human hematopoietic repopulation (see, FIG.7). This capacity is lost as the cells differentiate, marked byacquisition of CD45RA expression followed by CD38 expression and loss ofCD34 expression. These results show that surface phenotypes of thecorresponding markers varied widely among patients (Representative datafrom four patients are shown in FIG. 2). Population-level sequencingshowed patient-to-patient variation in mutations as expected. However,there were distinct differences in frequencies of mutated alleles amongpopulations with different cell surface phenotypes.

The transplanted patient-derived subpopulations were sorted according tosurface phenotypes into NSG mice. For example, in Patient 21 (FIG. 2A)and Patient 20 (FIG. 2B), CD34+CD38−subpopulation was further subdividedinto CD90−CD45RA− and CD90−CD45RA+subpopulations, corresponding toprimitive HSCs and more differentiated HPCs in normal humanhematopoiesis. Depending on the patient, subpopulations with similarsurface phenotypes showed distinct in vivo fates through NSGxenotransplantation. CD34+CD38−CD90−CD45RA−HSC-phenotype subpopulationfrom Patient 21 initiated AML in vivo while those from Patient 20 showedmultilineage engraftment. CD34+CD38−HSC/HPC-phenotype subpopulation fromPatient 13 (FIG. 2C) showed multilineage engraftment while those fromPatient 1 (FIG. 2D) initiated AML in vivo. In Patients 20 and 13,CD34+CD38−CD90−CD45RA+progenitor-phenotype and CD34−CD33+maturemyeloid-phenotype subpopulation contained LICs, respectively. Presenceand absence of FLT3−ITD mutation correlated with leukemia-initiation andmultilineage engraftment, respectively, while other mutations did notsegregate with in vivo cell fates.

Between patients, cells of the same surface phenotype nonetheless showeddistinct behaviors in vivo. For instance,CD34+CD38−CD90−CD45RA−population from Patient 21 initiated AML in NSGmice and therefore contained leukemia-initiating cells (LICs). On theother hand, population with the same phenotype population from Patient20 reconstituted multilineage human hematopoiesis in NSG mice andtherefore contained multilineage-engrafting hematopoietic stem cells.Similarly, Patient 13-derived CD34+CD38−population was amultilineage-engrafting hematopoietic stem cell-containing population,while Patient 1-derived CD34+CD38−population was a leukemia-initiatingcell-containing population.

Historically, differences in hematopoietic repopulation function havebeen attributed to cell surface phenotype, yet here, populations withthe same surface phenotype possessed different repopulating functionbetween patients. Thus, mutational profiling was performed at the singlecell level in each population as clonal diversity within a populationwith multiple mutations may not be characterized accurately by bulkallelic frequencies alone. Clonal structures within those subpopulationswere defined and mutation(s) associated with leukemia-initiatingfunction versus multilineage-engrafting function were identified at thesingle cell level.

Surface phenotype-defined leukemia-initiating cell-containingpopulations and multilineage-engrafting stem cell-containing populationsisolated from patients consisted of individual cells with diversecombinations of mutations (see, FIGS. 3A and 3B). For example, Patient21-derived CD34+CD38−CD90−CD45RA−single cells contained four distinctsubclones: FLT3−ITD+/DNMT3A-mutant, FLT3−ITD+/DNMT3A−WT, FLT3WT/DNMT3A-mutant or FLT3−WT/DNMT3A−WT (FIG. 3A). In addition,patient-to-patient variability in frequencies of mutations and theircombinations in individual cells were observed among bothleukemia-initiating cell-containing and multilineage-engrafting stemcell-containing populations. Every cell in Patient 1 leukemia-initiatingpopulation was FLT3−ITD+ whereas only 16 of 128 single cells wereFLT3−ITD+ in Patient 13 leukemia-initiating population (FIG. 3A).Similarly, every cell in Patient 13 multilineage-engrafting stem cellpopulation was FLT3−WT whereas 18 of 117 single cells were FLT3−ITD+ inPatient 20 multilineage-engrafting stem cell population (FIG. 3B). Thesefindings demonstrate that human AML cell subpopulations consisting ofsubclones with diverse mutational profiles have distinct in vivo cellfates (leukemia-initiating or multilineage-engrafting) when transplantedinto NSG recipients.

AML patient-derived leukemic and multilineage hematopoietic cellsengrafted in NSG recipients were also evaluated. By profiling mutationsin individual engrafted human leukemic cells and human multilineagehematopoietic cells, leukemia-initiating clones and pre-leukemic stemcell clones were identified among various subclones present inpatient-derived leukemia-initiating cell-containing andmultilineage-engrafting populations, respectively. Subsequently, bycomparing mutations present in individual clones with known in vivo cellfates, mutations that contribute to leukemia-initiation were determined.Surprisingly, in three cases (Patients 1, 13 and 21), every human cellin recipients transplanted with leukemia-initiating population wasFLT3−ITD+, regardless of the frequency of FLT3−ITD+single cells or thecell surface phenotype of the parental leukemia-initiating population.In addition, there were DNMT3A WT (Patients 1, 20 and 21), NPM1 WT(Patient 13) and TET2 WT (Patient 13) subclones among engraftedFLT3−ITD+AML cells in primary and secondary recipients, showing thatFLT3−ITD is most strongly linked with leukemogenesis and other mutationsare not obligatory (see, FIG. 3A).

This was not an observation limited to PDX models. In Patient 13, thefrequency of FLT3−ITD+single cells in leukemia-initiatingCD34−CD33+population substantially increased from initial diagnosis torelapse while NPM1 WT and TET2 WT subclones among the FLT3−ITD+subclonespersisted, showing that FLT−ITD conferred the greatest malignantpotential associated with relapse in the patient (see, FIG. 3A). On theother hand, every engrafted human cell in recipients transplanted withmultilineage-engrafting population was FLT3 WT, even in Patient 20 whosepre-leukemic (multilineage-engrafting) population contained FLT3−ITDsubclones (see, FIG. 3B). Moreover, there were DNMT3A-mutated (Patient21), NPM1-mutated (Patient 13) and TET2-mutated (Patient 13) subclonesin engrafted multilineage human hematopoietic cells in recipients,indicating that these mutations do not preclude multilineagehematopoiesis. Even single cells harboring both NPM1 and TET2 mutationsdifferentiated into CD19+B cells (see, FIG. 3B). These findings showthat FLT3−ITD mutation confers the greatest malignant potential withother mutations playing cooperative roles and strongly suggests thatacquisition of the FLT3−ITD mutation confers AML-initiating capacityregardless of surface phenotype and co-existing mutations.

Example 2

Effects of Kinase Inhibition on Human FLT3−ITD+AML Subclones withDiverse Mutations

These studies examined whether inhibition of a mutated kinase aloneeliminates AML in vivo using a NSG PDX model. Patient-derived cells witha FLT3−ITD mutation engraft in vivo and initiate AML, but the individualFLT3−ITD+cells harbor multiple other mutations in various combinations.These mutations may contribute to therapy responsiveness in cooperationwith FLT3−ITD, and a PDX model that reflects mutational complexity ofhuman AML cells is necessary to examine the contribution of multipleco-existing mutations. Therefore, the frequency of AML-associatedsomatic mutations was evaluated in the disclosed PDX model using newbornNSG xenotransplantation, and the same sets of mutations were present inpatient-derived leukemia-initiating population and engrafted AML cellsin recipients (see, FIG. 4). Moreover, single cell sequencingdemonstrated that the NSG PDX model allowed engraftment of multipleleukemia-initiating subclones with various combinations of mutations(see, FIGS. 3A and 3B). The extent that targeting FLT3 pathway alone canresult in reduction of human AML cells harboring multiple mutations invivo was examined as follows. RK-20449 is apyrrolo-pyrimidine-derivative multi-kinase inhibitor of Src familykinase HCK and FLT3 that effectively target human AML cells. Therefore,RK-20449 was administered to 58 NSG mice engrafted with AML from 19FLT3−ITD+AML patients and the results are shown in FIG. 8.

In five patients, responses were complete (every recipient treatedshowed residual

BM human CD45+chimerism <5%) (see, FIG. 5A). In these patients, RK-20449eliminated AML cells in the BM, spleen and PB to less than 0.01% basedon flow cytometry and immunohistochemistry, such as about 0.05% to about0.01%. in all recipients treated, despite the presence of multiplemutations not directly targeted by RK-20449 (Patient 1: DNMT3A, CEBPA,TET2; Patient 2: IDH1; Patient 15: CEBPA, n-Ras; Patient 16: WT1). In 11additional cases, RK-20449 alone resulted in complete responses in thespleen in a majority of recipients tested (see, FIG. 5B). Overall, inall 19 cases tested, FLT3−ITD+human AML cells show significant responsesto single agent RK-20449 in vivo; however, residual AML cells wereobserved in the BM of at least one treated mouse in 14 of 19 cases (see,FIGS. 5C and 5D). Therefore, mechanisms for multi-kinase inhibitionresistance in FLT3−ITD+AML cells were evaluated.

Example 3

Effect of Combined Inhibition of Kinase and Anti-Spoptosis Pathways onFLT3−ITD+AML Cells in Vivo

Recipients engrafted with AML from cases in which kinase inhibitionalone did not completely eradicate human AML cells in the BM and spleenwere treated with RK-20449 combined with ABT-199. See, FIGS. 9-11, fordata and statistics. PB hCD45+AML cell chimerism time-course for treatedrecipients illustrate the differences in the rate and degree ofperipheral responses associated with type of treatment (see, FIG. 6A).In the majority of cases, apoptosis induction by BCL-2 inhibitor ABT-199alone resulted in limited responses. In contrast, in all 12 cases,combination treatment significantly reduced human AML chimerisms in PB,BM and spleen. Moreover, in 9 of 12 cases, combined inhibition of kinaseand anti-apoptosis pathways led to elimination of human AML cells invivo below the limit of detection without targeting other co-existingmutations (see, FIGS. 6B and 8).

Residual viable human AML cells were too few to assess functionally inmost recipients that showed complete responses to RK-20449/ABT-199combination treatment. To assess the effect of combination treatment toAML stem cells, human AML cells were isolated from cases that showedcomplete responses, but with relatively high residual BM human CD45chimerisms, serial transplantation into NSG mice was performed (see,FIG. 6C). Leukemia-initiating capacity remaining after treatment wascompared by isolating residual viable human CD45+AML cells from eachtreated mouse and transplanting a pre-determined fraction into secondaryNSG recipients. The RK-20449 alone- and ABT-199 alone-treated BMcontained enough viable LSCs to initiate AML in every secondaryrecipient transplanted. In contrast, among 23 mice transplanted withresidual AML cells from recipients treated with combination treatment,only one engrafted, indicating that combination treatment with RK-20449and ABT-199 more effectively reduced the frequency of LICs in vivo.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of co-inhibiting HCK and BCL-2 in a cell, comprisingcontacting the cell with an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.
 2. A method of killinga cell having an FLT3−ITD mutation, comprising contacting the cell withan HCK inhibitor and a BCL-2 inhibitor, or a pharmaceutically acceptablecomposition thereof.
 3. The method of claim 1 or 2, further comprisingcontacting the cell with an FLT3−ITD inhibitor.
 4. The method of claim 1or 2, wherein the HCK inhibitor is a dual HCK/FLT3−ITD inhibitor.
 5. Amethod of treating acute myeloid leukemia, comprising conjointlyadministering to a subject an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.
 6. The method of claim5, wherein the subject has FLT3−ITD+acute myeloid leukemia.
 7. Themethod of claim 5 or 6, wherein the subject has malignant hematopoiesisand/or non-malignant multilineage hematopoiesis characterized by cellshaving one or more mutations in a gene selected from DNMT3A, IDH2, IDH1,NPM1, TET2, CEBPA, ASXL1, EZH2, SETBP1, SMC3, KIT, NRAS, and WT1.
 8. Themethod of any one of claims 5-7, further comprising conjointlyadministering a FLT3−ITD inhibitor.
 9. The method of any one of claims5-8, wherein the HCK inhibitor is a dual HCK/FLT3−ITD inhibitor.
 10. Themethod of claim 8 or 9, wherein the HCK inhibitor, the FLT3−ITDinhibitor, and the BCL-2 inhibitor are administered simultaneously orsequentially in separate unit dosage forms.
 11. The method of any one ofclaims 5-10, comprising administering a single unit dosage formcomprising an HCK inhibitor, a BCL-2 inhibitor, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.
 12. The method of claim 11,wherein the single unit dosage form further comprises an FLT3−ITDinhibitor, or wherein the HCK inhibitor is a dual HCK/FLT3−ITDinhibitor.
 13. The method of any preceding claim, wherein the HCKinhibitor is selected from RK-20449, RK-20693, RK-24466, RK-20444,RK-20445, and RK-20466.
 14. The method of any one of claim 3, 4, or7-12, wherein the FLT3−ITD inhibitor is selected from AC220, sorafenib,PKC412, CEP-701, UNC2025, MLN518, KW-2449, AMG-925, sunitinib, SU5614,AC2206, crenolanib, and PLX3397.
 15. The method of any preceding claim,wherein the BCL-2 inhibitor is selected from AT-101, TW-37, TM-1206,gossypolic acid, gossypolonic acid, apogossypol, apogossypolone,A385358, ABT-737, ABT-263, ABT-199, WEHI-539, BXI-61, BXI-72, obatoclax,JY-1-106, and SAHB peptides.
 16. The method of claim 15, wherein theBCL-2 inhibitor is selected from gossypol, obatoclax, ABT-737, ABT-199,and ABT-263.
 17. The method of claim 16, wherein the BCL-2 inhibitor isABT-199.
 18. The method of claim 17, wherein the HCK inhibitor isRK-20449 and the BCL-2 inhibitor is ABT-199.
 19. The method of claim 17,wherein the HCK inhibitor is RK-20693 and the BCL-2 inhibitor isABT-199.
 20. The method of claim 17, wherein the FLT3−ITD inhibitor isAC220 and the BCL-2 inhibitor is ABT-199.
 21. The method of claim 16,wherein the FLT3−ITD inhibitor is SU5614 and the BCL-2 inhibitor isABT-737.
 22. The method of any preceding claim, wherein the HCKinhibitor, and/or FLT3−ITD inhibitor, and/or the BCL-2 inhibitor is eachpresent as a pharmaceutically acceptable salt.
 23. The method of anypreceding claim, wherein the HCK inhibitor, and/or FLT3−ITD inhibitor,and/or the BCL-2 inhibitor is each present in a pharmaceuticallyacceptable composition.
 24. A composition for co-inhibiting HCK andBCL-2, comprising an HCK inhibitor and a BCL-2 inhibitor, or apharmaceutically acceptable composition thereof.
 25. A composition forkilling a cell having an FLT3−ITD mutation, comprising an HCK inhibitorand a BCL-2 inhibitor, or a pharmaceutically acceptable compositionthereof.
 26. The composition of claim 24 or 25, further comprisingcontacting the cell with an FLT3−ITD inhibitor.
 27. The composition ofclaim 24 or 25, wherein the HCK inhibitor is a dual HCK/FLT3−ITDinhibitor.
 28. A composition for treating acute myeloid leukemia,comprising conjointly administering to a subject an HCK inhibitor and aBCL-2 inhibitor, or a pharmaceutically acceptable composition thereof.29. The composition of claim 28, wherein the subject has FLT3−ITD+acutemyeloid leukemia.
 30. The composition of claim 28 or 29, wherein thesubject has malignant hematopoiesis and/or non-malignant multilineagehematopoiesis characterized by cells having one or more mutations in agene selected from DNMT3A, IDH2, IDH1, NPM1, TET2, CEBPA, ASXL1, EZH2,SETBP1, SMC3, KIT, NRAS, and WT1.
 31. The composition of any one ofclaims 28-30, further comprising conjointly administering a FLT3−ITDinhibitor.
 32. The composition of any one of claims 28-31, wherein theHCK inhibitor is a dual HCK/FLT3−ITD inhibitor.
 33. The composition ofclaim 31 or 32, wherein the HCK inhibitor, the FLT3−ITD inhibitor, andthe BCL-2 inhibitor are administered simultaneously or sequentially inseparate unit dosage forms.
 34. The composition of any one of claims28-33, comprising administering a single unit dosage form comprising anHCK inhibitor, a BCL-2 inhibitor, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.
 35. The composition of claim 34, whereinthe single unit dosage form further comprises an FLT3−ITD inhibitor, orwherein the HCK inhibitor is a dual HCK/FLT3−ITD inhibitor.
 36. Thecomposition of any preceding claim, wherein the HCK inhibitor isselected from RK-20449, RK-20693, RK-24466, RK-20444, RK-20445, andRK-20466.
 37. The composition of any one of claim 26, 27, or 30-35,wherein the FLT3−ITD inhibitor is selected from AC220, sorafenib,PKC412, CEP-701, UNC2025, MLN518, KW-2449, AMG-925, sunitinib, SU5614,AC2206, crenolanib, and PLX3397.
 38. The composition of any precedingclaim, wherein the BCL-2 inhibitor is selected from AT-101, TW-37,TM-1206, gossypolic acid, gossypolonic acid, apogossypol,apogossypolone, A385358, ABT-737, ABT-263, ABT-199, WEHI-539, BXI-61,BXI-72, obatoclax, JY-1-106, and SAHB peptides.
 39. The composition ofclaim 38, wherein the BCL-2 inhibitor is selected from gossypol,obatoclax, ABT-737, ABT-199, and ABT-263.
 40. The composition of claim39, wherein the BCL-2 inhibitor is ABT-199.
 41. The composition of claim40, wherein the HCK inhibitor is RK-20449 and the BCL-2 inhibitor isABT-199.
 42. The composition of claim 40, wherein the HCK inhibitor isRK-20693 and the BCL-2 inhibitor is ABT-199.
 43. The composition ofclaim 40, wherein the FLT3−ITD inhibitor is AC220 and the BCL-2inhibitor is ABT-199.
 44. The composition of claim 39, wherein theFLT3−ITD inhibitor is SU5614 and the BCL-2 inhibitor is ABT-737.
 45. Thecomposition of any preceding claim, wherein the HCK inhibitor, and/orFLT3−ITD inhibitor, and/or the BCL-2 inhibitor is each present as apharmaceutically acceptable salt.
 46. The composition of any precedingclaim, wherein the HCK inhibitor, and/or FLT3−ITD inhibitor, and/or theBCL-2 inhibitor is each present in a pharmaceutically acceptablecomposition.